1. Field of the Invention
We herein describe the formulation of a reagent which can be used as a marker to identify individual lanes on an electrophoresis gel which is run on an Automated DNA Sequencer. This reagent is intended to be included in the loading dye for samples in at least the following applications: DNA sequencing, genetic fragment analysis, linkage mapping analysis, human DNA identity testing, animal DNA identity testing, and DNA paternity testing. Inclusion of the reagent aids in the identification of the lane in which each sample is individually loaded on the acrylamide gel, such that the data read from each lane by the instrument is correctly applied to the appropriate sample. Specifically, this invention is the formulation of a fluorescent dye label attached to a synthetic DNA molecule (oligonucleotide) which has been constituted in a loading buffer. The invention will facilitate manual lane tracking of all samples loaded on an automated DNA sequencer acrylamide gel. With the appropriate modifications to the existing software, he invention will facilitate automated lane tracking on DNA sequencer acrylamide gels.
2. Description of the Related Art
Development of rapid and sensitive nucleic acid sequencing methods utilizing automated fluorescence detection strategies has revolutionized modern molecular biology (see, e.g., Sanger, F., S. Nicklen, A. R. Coulson (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467; Kaiser, R. J., et al. (1989) Nucl. Acids. Res. 17(15):6087-6102; Hunkapillar, T. et al. (1991) Science 254(5028):59-67). Analysis of entire genomes of plants, fungi, animals, bacteria, and viruses is now possible. To keep pace with the huge volume of data required to decipher the genome of any particular organism, modern sequencing instruments have been developed which allow for high throughput sample analysis while automating sample handing and data analysis. However, there continue to be technical problems for accurate sample identification throughout the sequencing process.
One technical problem which hinders sequence analysis of DNA is the inability of the investigator or the DNA sequencer to accurately monitor sample application "lanes" on DNA sequencing acrylamide gels. For example, it is not at all uncommon for samples which have been reconstituted with the standard loading dye formulation to contain no fluorescent components (due to failures of the sequencing reaction chemistries) or to contain only moderately fluorescent sequencing components, due to partial failures of the sequencing reaction chemistries.
Prior to applying the samples to the lanes of a sequencing acrylamide gel, however, the investigator has no idea whether a particular sample reaction has been successful. Imagine, then, that some unsuccessful reaction samples are loaded in the midst of successful reaction samples across 96 lanes of a sequencing gel. As a consequence, improper lane assignments (called tracking) are often made because the automated sequencer fails to mark (i.e., skips) lanes which do not contain fluorescent components. This critical mistake often leads to improper lane assignments which in turn often adversely affects al subsequent data analysis, including misinterpretation of the DNA sequence of the sample under study.
Accordingly, it is clear that there exists a need in the art for proper lane identification and assignment, regardless of the success outcome of the sequencing chemistry reaction. Use of such a method would inevitably improve the results of high throughput screening DNA sequence analysis.